Fastsybr mixture with rox

RNA isolation and qPCR were performed as described previously62 (link). Briefly, PAMs were infected with PRRSV at an MOI of 0.1 for 24 h, and then treated with or without poly(I:C) (10 μg/ml) for 6 h. Total RNAs were extracted with TRIzol (Invitrogen) and used for cDNA synthesis using M-MLV reverse transcriptase according to the manufacturer’s instructions (Takara). Quantitative RT-PCR (qPCR) analysis was performed using FastSYBR Mixture with ROX (Cwbiotech) on the ViiA^TM7 real-time PCR System (Applied Biosystems). Gene-specific primers for Mx1, OAS, VISA and GAPDH were designed and listed in Table 1. The expression of Mx1 and OAS was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as fold induction relative to the control. VISA expression was normalized to the mock infection.

RNA was extracted with TRIzol reagent (Invitrogen Life Technology) following the manufacturer’s instruction. RNA (1 μg) was then reverse-transcribed to cDNA using M-MLV reverse transcriptase according to the manufacturer’s instructions (Takara). Quantitative real-time PCR (qRT-PCR) analysis was performed using FastSYBR Mixture with ROX (Cwbiotech) on the ViiA™7 qRT-PCR System (Applied Biosystems). Gene-specific primers for GAPDH, COX-1, COX-2, and ORF7 were listed in Table 4.